Method for the direct diagnostic detection of genetically caused pathogenic point mutations

ABSTRACT

The present invention relates to a quick method for the qualitative and quantitative medical-diagnostic analysis on the protein level of the substitution of single amino acids with pathogenic and non-pathogenic effects on the organism. The medical-diagnostic analysis is performed by a combination of enzymatic or chemical cleavage of the isolated peptide, chromatographical separation of the fragments and analysis by mass spectrometry, both direct LC/MS and indirect MALDI-MS, and analysis by capillary electrophoresis. By comparing protein samples from healthy humans with those of ill humans, the method described is suitable for establishing new, as yet unknown mutations and quantifying the expression and incorporation of wild type to mutant.

The present invention relates to a method according to the generic partof claim 1.

Mutations on the gene level often cause inappropriate amino acids to beincorporated in proteins which are encoded in the corresponding genesegment which has mutated. This may result in pathologic phenomena,e.g., sickle-cell anaemia. Examinations of the presence of mutationswhich are responsible for pathologic conditions are usually performed onthe DNA level. Such examinations are cumbersome, and their evaluationtakes very long as a rule.

Thus, an object of the invention has been to provide a method by whichthe presence of mutations can be established quickly and reliably.

According to the invention, this object is achieved by a method with thefeatures as defined in claim 1.

The method according to the invention serves to recognize mutations inorganisms by comparing a deviation, Caused by a mutation, of the aminoacid composition of a protein expressed in the region of the mutation(protein to be examined) with a corresponding protein which is expressedby a wild type lacking the mutation. The method according to theinvention is characterized in that

a sample is taken from the organism at a site where the protein to beexamined is expressed, can be detected and/or plays a physiologicalrole;

either the protein is concentrated or purified by methods of proteinanalysis, followed by a determination of its molecular weight, or

a determination of the molecular weight of the protein to be examined isperformed without a pretreatment of the sample.

The invention advantageously relates to a quick method for a qualitativeand quantitative medical-diagnostic analysis on the protein level of thesubstitution of single amino acids with pathogenic or non-pathogeniceffects on the organism. The medical-diagnostic analysis is preferablyperformed by a combination of enzymatic or chemical cleavage of theisolated peptide, chromatographical separation of the fragments andanalysis by mass spectrometry, both direct LC/MS and indirect MALDI-MS,and analysis, e.g., by capillary electrophoresis. By comparing proteinsamples from healthy subjects with those of ill subjects, the methoddescribed is suitable for establishing new, as yet unknown mutations andquantifying the expression and incorporation of wild type to mutant.

The obtaining of samples from an organism, especially by biopsy, can beconsidered a basis of the method according to the invention. As thesites where the samples are taken, sites are selected in which theprotein to be examined is expressed, detectable or plays a physiologicalrole, e.g., in muscle fiber bundles or tissue pieces from organs,especially hearts. The samples are advantageously processed in such away that the substitution of a single amino acid can be unambiguouslydetected by mass-spectrometric, chromatographical and/orelectrophoretical methods, and the expression and incorporation ratio ofwild type to mutant can be quantitatively determined. Thus, it becomespossible by a direct protein analysis as well to diagnose the causes ofa genetically caused disease at an early stage and to identify as yetunknown mutations by a comparative analysis of samples from healthysubjects and samples from ill subjects.

By using the highly sensitive “matrix-assisted laser-induced desorptionand ionization time-of-flight mass spectrometry” (MALDI-MS) incombination with liquid chromatography, especially using columns with aninner diameter of ≦1 mm (microbore and capillary columns), the detectioncan be effected even with minute sample quantities.

As compared to the as yet performed analyses of the DNA coding for theproteins, the method is characterized in that the detection of themutation and the determination of the expression and incorporation ratiois effected in a significantly shorter period of time. An analysis canbe performed within one week, in contrast to the usual times requiredfor DNA analysis, ranging from several weeks to half a year.

It is an advantage that a specific cleavage of the protein recoveredfrom biopsy samples for diagnostic purposes is performed with selectiveenzymes, endoproteinases, or by chemical reagents, and the fragmentsobtained by such cleavage are separated by chromatographical methods andcharacterized in terms of their molecular weights either directly(LC/MS) or indirectly (MALDI-MS). The distribution in the separation andthe molecular weight, determined by mass spectrometry, is unambiguousevidence for each fragment of the protein.

With smaller proteins, i.e., those having a molecular weight of up to100,000 Dalton, the amino acid substitutions can be directly determinedwith the method described, without a preliminary cleavage intofragments. The accuracy of the measurement enables the precisedetermination of deviations in molecular weight of <5 Dalton and thusthe detection of both wild type and mutant in the presence of eachother. The precise localization of the amino acid substitution can beeffected by the above described cleavage.

Thus, it is possible to characterize all proteins involved in aphysiologically functional structure, e.g., muscle, in terms ofgenetically caused pathogenic and non-pathogenic amino acidsubstitutions.

Since the amino acid sequences of a lot of proteins are already knowndue to the sequencing of the human genome and all human proteins will beelucidated in a few years, a protein can be analyzed for mutations bymeans of the method herein described by selecting the appropriatecleavage which can be achieved by one skilled in the art by per se knownmethods.

It can be of great importance that this method may also be adapted forclinical-diagnostic use in the recognition of the causes of diseasesand, through an optimization of the separation and detection conditions,also for the systematic screening of patient samples for particular,defined mutations.

Using appropriate equipment, the method described may also be automatedto a large extent. For the procedure as described, the duration of ananalysis is in the range of a few days whereas DNA analysis takesseveral weeks to months. At the same time, all proteins involved in aphysiologically functional structure can be analyzed by this method.

Unlike the conventional methods for the detection of point mutations byanalyzing the genomic DNA or cDNA, this method allows the quantificationof the expression and incorporation of the mutant in the physiologicallyfunctional structure, especially if, due to the presence of a diploidset of chromosomes in the nucleus, both forms of the respective proteinare expressed and it is not known in what ratio the two forms arepresent in the functional form.

The invention further allows evidence of the identity of the wild typeand of the mutant to be furnished by chemical sequence analysis or byamino acid analysis.

A preferred embodiment of the method according to the inventioncomprises the cleavage of the protein to be examined with suitableenzymes or chemical reagents, and a combination of chromatographicalmethods, especially high-pressure liquid chromatography with capillarycolumns (MB-HPLC), and mass spectrometry.

The invention will be further illustrated by using the heavy chain ofthe β-isoform of myosin as an example. Point mutations in the heavychain of the ⊖-isoform of myosin, e.g., substitution of the amino acidmethionine for the amino acid valine in position 606, may result inhypertrophic cardiomyopathy, a genetically caused thickening of certainheart walls which may lead to sudden death. According to the invention,the detection of the mutation is possible by a combination of enzymaticcleavage and LC/MS.

FIGS. 1a and 1 b show the analysis of the peptide fragments of humancardiac β-myosin heavy chain (β-MHC) by means of a coupling of highperformance liquid chromatography (HPLC) with mass spectrometry (MS). Inthis example, the presence of the heterozygotic mutant Val606Met, i.e.,substitution of valin in position 606 of β-MHC, was looked for. FIG. 1ashows two marked ranges in which the fragment with a substituted aminoacid and a molecular weight of 1507.5 could be detected in addition tothe original fragment, the wild type fragment having a molecular weightof 1475.5, in a person for whom the substitution had been proven on thegene level. For comparison, FIG. 1b shows the analysis of a β-MHC sampleof a person with no point mutation in this gene. In this case, only thewild type fragment can be detected; in the range in which the mutatedfragment was eluted in FIG. 1a, this peptide is completely lacking.Thus, it becomes possible to detect mutated β-MHC in a person's musclefibers.

In order to support this finding which is only substantiated by theobserved shift in molecular weight of +32 by the amino acid substitutionof Val by Met, fragments of an enzymatic cleavage were separated byHPLC, and the fractions were examined for the presence of peptides withmolecular weights of 1475.5 and 1507.5 using mass spectrometry.

These peptides could be found in such fractions as corresponded to theabove showed LC/MS analysis. FIGS. 2a and 2 b show the mass spectra ofthese peptide fractions; in addition to the peptides to be detected, alarge number of other peptides from β-MHC are also present.

The sequences of the peptides detected in the fractions were confirmedby collision-induced fragmentation by means of the coupling of two massspectrometers (CID-MS/MS). The distances between the fragment ionsdesignated as b and y correspond to the amino acids of the peptides withhigh specificity. From these distances, the sequence of the peptide canbe established fully or in part. Problems are only encountered with theamino acids Leu and Ile which have no difference in molecular weight atall, and Lys and Gln which are different by only 0.04 unit masses. Sincethe enzymatic cleavage was performed with an enzyme which specificallycleaves behind Lys, the sequence determination can be based on theassumption that amino acids at the end of the peptide are Lys.

The CID-MS/MS spectra are shown in FIGS. 3a and 3 b. The distancebetween b5 and b6 clearly shows the shift caused by the amino acidsubstitution while all other distances between the fragment ions remainunchanged. The evaluation yielded DPLNETVVGLYQK (SEQ ID No. 1) as thesequence for the wild type, and DPLNETVMGLYQK (SEQ ID No. 2) as thesequence for the mutant. Comparisons performed in data bases yielded nohomology of the CID-MS/MS spectra with other proteins, except for veryhomologous β-MHCs from other species. Thus, the presence of the mutationin the muscle fibers could be clearly proven on a molecular level.

The stated techniques are not suitable for quantifying the degree of theincorporation of wild type to mutant. Because of the differentionization probabilities and the interference by other peptides presentin the mixtures, the amount of peptide cannot be concluded from theresults of mass spectrometry. For a quantification, another separationmethod is recommendable as a quantitative detection method, namelycapillary zone electrophoresis. In this method which is based on otherspecific parameters than those of HPLC, peptides are separated in.unmodified glass capillaries, filled with buffers, with an innerdiameter of 0.05 to 0.075 mm and a length of 50 cm in a strong electricfield. The buffer employed has a pH value of 2.5 (100 mM phosphatebuffer) in order to exclude electroendoosmotic fluxes of the bufferitself and to confer a positive charge to all peptides. In this system,the peptides of the identified fractions were separated. A commerciallyavailable peptide (low-pH mobility marker of Applied Biosystems) wasadded in the same amounts as an internal standard, in order to be ableto compare, on one hand, the peak areas of the peptides to be compared,and to consider variations in the elution time in the different runs.The peptides to be detected themselves were identified in furtherseparations by coinjection with a synthetic peptide of the samesequence. From the peak height and also from the peak area, a relativeincorporation of the mutant of 10 to 15% of the total β-MHC wascalculated. This experiment was performed with 5 different preparations,but from one patient. The thus measured values for the mutant β-MHVVal606Met were always between 10 and 15%. The capillary zoneelectrophoreses are shown in FIGS. 4a and 4 b, the marked peptide (*) isthe added internal standard, peptide (1) is the fragment of the wildtype of the β-MHC, and peptide (2) is the fragment of the mutant of theβ-MHC.

It is interesting that the sample examined does not show any symptoms ofthe familial hypertrophic cardiomyopathy. Therefore, it can beconsidered that a low degree of incorporation does not result inexternally visible changes in muscle function.

The method according to the invention can-generally be applied to allmutants of the β-MHC. As yet, several fragments which are potentialcarriers of mutations were successfully identified; these fragments areunderlined in FIG. 5, and the amino acids written below the continuoussequence are the as yet known substitutions. The method according to theinvention can further be applied to all mutations having a pathogeniceffect. For diagnostic purposes, this method may be used for the earlyrecognition of the development of hereditary diseases;. in addition togene analysis, a direct forecast of the risk of a severe disease or ofthe course of the disease can be made for every patient by analyzing thefunctioning proteins from the organism.

Example 1

LC/MS analysis of fragments of the heavy chain of the β-isoform ofmyosin (β-MHC) obtained with endoproteinase Lys-C

The basis of the selective cleavage is the isolation of the β-MHC frommuscle fibers of the soleus muscle which are obtained from biopsies bydissolution in a high salt buffer and precipitation of the β-MHC bydecreasing the salt concentration. The collected β-MHC may be washedwith water to remove disturbing soluble components.

For preparing the fragments to be analyzed, the precipitated β-MHC isslurried in 200 μl of 100 mM ammonium .hydrogencarbonate buffer, pH 8.2,and incubated with endoproteinase Lys-C (Boehringer Mannheim) at 37° C.for more than 24 hours. The solution is then subjected to freeze-drying,and the residue is dissolved in 100 μl of 0.1 M trifluoroacetic acid inwater.

To remove undissolved components, the sample is filtered through an 0.2μm cellulose acetate filter at a maximum of 10,000×g, and 20 to 50 μlaliquots are applied on the MB-HPLC column. The separation is performedthrough a reverse phase C18 column with an inner diameter of 1 mm and alength of 100 mm at a flow rate of 20 μl/min. The elution of thepeptides is effected by a linear increase of 0.5%/min of theacetonitrile concentration in the buffer, from 10% acetonitrile in 0.06%TFA in water to 80% acetonitrile in 0.05% TFA in water. The elutingpeptides are directed through a fused silica capillary and a UV detectordirectly into the electrospray ionization device of the quadrupole massspectrometer (SCIEX API III, Perkin-Elmer, Langen). In the massspectrometer, the m/z (mass to charge) values are determined over arange of from 400 to 2400 amu (atomic mass units) every 4.2 seconds.From the recorded total ion chromatogram and the respective massspectra, the fragment of the wild type and of the mutant can be detectedby computer aided evaluation.

After the separation described, a sample of the non-mutated β-MHC wasanalyzed together with the synthetic peptides having the sequences ofthe non-mutated and mutated fragments under the same conditions. Themolecules which had been found are in the ranges in which the syntheticpeptides are eluted.

FIG. 1: Top part: Total ion chromatogram of the separation of thefragments of the mutated β-MHC obtained with endoproteinase Lys-C.

Middle part: Detection of the non-mutated fragment in the cross-hatchedrange (1) of the top figure. The non-mutated fragment has a molecularweight of 1475.5 amu.

Bottom part: Detection of the mutated fragment in the crosshatched range(2) of the top figure. The mutated fragment has a molecular weight of1507.5 amu.

EXAMPLE 2

Detection of the fragments of the wild type and the mutant of the β-MHCby means of the MALDI-MS technique

As described in Example 1, the cleavage of the β-MHC was effected byincubation with endoproteinase Lys-C and a subsequent separation on areverse phase C18 column under the conditions stated above. In thisExample, instead of direct coupling with the mass spectrometer, anautomated fraction collector collects fractions for 2 minutes each, and0.5 μl each of these fractions is mixed with 1 μl of a suitable matrixsubstance, usually α-hydroxycinnamic acid or sinapic acid, dissolved in60% acetonitrile and 0.2% TFA, on a support for MALDI-MS. Thedetermination of the molecular weights is then performed in a MALDI-MS(Vestec, Houston) using an external standard, i.e., two known syntheticpeptides, for calibrating the measurement.

In the corresponding fractions from Example 1, these measurementsyielded the expected masses of 1477.5 and 1507.5 amu.

Thus, the mutated fragments can be unambiguously detected by means ofMALDI-MS as well.

EXAMPLE 3

Analysis of the fractions from Example 2 by means of capillaryelectrophoresis

The fractions from Example 2 were separated by capillary electrophoresis(Beckmann P/ACE 2000, Beckmann, Munich) using fused silica capillarieswith an inner diameter of 75 μm in a 100 mM sodium phosphate buffer, pH8.0, at a voltage of 15 to 17 kV. The detection of the peptides waseffected by means of a UV detector at 200 nm.

By coinjecting the synthetic peptides in a second separation run, theidentity of the natural non-mutated and mutated fragments could beshown.

3 1 1120 PRT Homo sapiens MOD_RES (26) “Xaa” represents Ala or Val 1 MetGly Asp Ser Glu Met Ala Val Phe Gly Ala Ala Ala Pro Tyr Leu 1 5 10 15Arg Lys Ser Glu Lys Glu Arg Leu Glu Xaa Gln Thr Arg Pro Phe Asp 20 25 30Leu Lys Lys Asp Val Phe Val Pro Asp Asp Lys Gln Glu Phe Val Lys 35 40 45Ala Lys Ile Val Ser Arg Glu Gly Gly Lys Xaa Thr Ala Glu Thr Glu 50 55 60Tyr Gly Lys Thr Val Thr Val Lys Glu Asp Gln Val Met Gln Gln Asn 65 70 7580 Pro Pro Lys Phe Asp Lys Ile Glu Asp Met Ala Met Leu Thr Phe Leu 85 9095 His Glu Pro Ala Val Leu Tyr Asn Leu Lys Asp Arg Tyr Gly Ser Trp 100105 110 Met Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Thr Val Asn Pro Tyr115 120 125 Lys Trp Leu Pro Val Tyr Thr Pro Glu Val Val Ala Ala Tyr XaaGly 130 135 140 Lys Lys Arg Ser Glu Ala Pro Pro His Ile Phe Ser Ile SerAsp Asn 145 150 155 160 Ala Tyr Gln Tyr Met Leu Thr Asp Arg Glu Asn GlnSer Ile Leu Ile 165 170 175 Thr Gly Glu Ser Gly Ala Gly Lys Thr Val AsnThr Lys Arg Val Ile 180 185 190 Gln Tyr Phe Ala Val Ile Ala Ala Ile GlyAsp Arg Ser Lys Lys Asp 195 200 205 Gln Ser Pro Gly Lys Gly Thr Leu GluAsp Gln Ile Ile Gln Ala Asn 210 215 220 Pro Ala Leu Glu Ala Phe Gly AsnAla Lys Thr Val Arg Asn Asp Asn 225 230 235 240 Ser Ser Arg Phe Gly LysPhe Ile Xaa Ile His Phe Gly Ala Thr Xaa 245 250 255 Lys Leu Ala Ser AlaAsp Ile Glu Thr Tyr Leu Leu Glu Lys Ser Arg 260 265 270 Val Ile Phe GlnLeu Lys Ala Glu Arg Asp Tyr His Ile Phe Tyr Gln 275 280 285 Ile Leu SerAsn Lys Lys Pro Glu Leu Leu Asp Met Leu Leu Ile Thr 290 295 300 Asn AsnPro Tyr Asp Tyr Ala Phe Ile Ser Gln Gly Glu Thr Thr Val 305 310 315 320Ala Ser Ile Asp Asp Ala Glu Glu Leu Met Ala Thr Asp Asn Ala Phe 325 330335 Asp Val Leu Gly Phe Thr Ser Glu Glu Lys Asn Ser Met Tyr Lys Leu 340345 350 Thr Gly Ala Ile Met His Phe Gly Asn Met Lys Phe Lys Leu Lys Gln355 360 365 Arg Glu Glu Gln Ala Glu Pro Asp Gly Thr Glu Glu Ala Asp LysSer 370 375 380 Ala Tyr Leu Met Gly Leu Asn Ser Ala Asp Leu Leu Lys GlyLeu Cys 385 390 395 400 His Pro Xaa Val Lys Val Gly Asn Glu Tyr Val ThrLys Gly Gln Asn 405 410 415 Val Gln Gln Val Ile Tyr Ala Thr Gly Ala LeuAla Lys Ala Val Tyr 420 425 430 Glu Arg Met Phe Asn Trp Met Val Thr ArgIle Asn Ala Thr Leu Glu 435 440 445 Thr Lys Gln Pro Xaa Gln Tyr Phe IleGly Val Leu Asp Ile Ala Gly 450 455 460 Phe Glu Ile Phe Asp Phe Asn SerPhe Glu Gln Leu Cys Ile Asn Phe 465 470 475 480 Thr Asn Glu Lys Leu GlnGln Phe Phe Asn His His Met Phe Val Leu 485 490 495 Glu Gln Glu Glu TyrLys Lys Glu Gly Ile Glu Trp Thr Phe Ile Asp 500 505 510 Xaa Gly Met AspLeu Gln Ala Cys Ile Asp Leu Ile Glu Lys Pro Met 515 520 525 Gly Ile MetSer Ile Leu Glu Glu Glu Cys Met Phe Pro Lys Ala Thr 530 535 540 Asp MetThr Phe Lys Ala Lys Leu Phe Asp Asn His Leu Gly Lys Ser 545 550 555 560Ala Asn Phe Gln Lys Pro Arg Asn Ile Lys Gly Lys Pro Glu Ala His 565 570575 Phe Ser Leu Ile His Tyr Ala Xaa Ile Val Xaa Tyr Asn Ile Ile Gly 580585 590 Trp Leu Gln Lys Asn Lys Asp Pro Leu Xaa Glu Thr Val Xaa Gly Leu595 600 605 Tyr Gln Lys Ser Ser Leu Xaa Leu Leu Ser Thr Leu Phe Ala AsnTyr 610 615 620 Ala Gly Ala Asp Ala Pro Ile Glu Lys Gly Lys Gly Lys AlaLys Lys 625 630 635 640 Gly Ser Ser Phe Gln Thr Val Ser Ala Leu His ArgGlu Asn Leu Asn 645 650 655 Lys Leu Met Thr Asn Leu Arg Ser Thr His ProHis Phe Val Arg Cys 660 665 670 Ile Ile Pro Asn Glu Thr Lys Ser Pro GlyVal Met Asp Asn Pro Leu 675 680 685 Val Met His Gln Leu Arg Cys Asn GlyVal Leu Glu Gly Ile Arg Ile 690 695 700 Cys Arg Lys Gly Phe Pro Asn ArgIle Leu Tyr Xaa Asp Phe Xaa Gln 705 710 715 720 Arg Tyr Xaa Ile Leu AsnPro Ala Ala Ile Xaa Glu Gly Gln Phe Xaa 725 730 735 Asp Ser Arg Lys XaaAla Glu Lys Leu Leu Ser Ser Leu Asp Ile Asp 740 745 750 His Asn Gln TyrLys Phe Gly His Thr Lys Val Phe Phe Lys Ala Gly 755 760 765 Leu Leu GlyLeu Leu Glu Glu Met Arg Xaa Glu Arg Leu Ser Arg Ile 770 775 780 Ile ThrArg Ile Gln Ala Gln Ser Arg Gly Val Leu Xaa Arg Met Glu 785 790 795 800Tyr Lys Lys Leu Leu Glu Arg Arg Asp Ser Leu Leu Val Ile Gln Trp 805 810815 Asn Ile Arg Ala Phe Met Gly Val Lys Asn Trp Pro Trp Met Lys Leu 820825 830 Tyr Phe Lys Ile Lys Pro Leu Leu Lys Ser Ala Glu Arg Glu Lys Glu835 840 845 Met Ala Ser Met Lys Glu Glu Phe Thr Arg Leu Lys Glu Ala LeuGlu 850 855 860 Lys Ser Glu Ala Arg Xaa Lys Glu Leu Glu Glu Lys Met ValSer Leu 865 870 875 880 Leu Gln Glu Lys Asn Asp Leu Gln Leu Gln Val GlnAla Glu Gln Asp 885 890 895 Asn Leu Ala Asp Ala Glu Glu Arg Cys Asp GlnXaa Ile Lys Asn Lys 900 905 910 Ile Gln Leu Glu Ala Lys Val Lys Glu MetAsn Xaa Arg Leu Glu Asp 915 920 925 Glu Glu Glu Met Asn Ala Xaa Leu ThrAla Lys Lys Arg Lys Leu Glu 930 935 940 Asp Glu Cys Ser Xaa Leu Lys ArgAsp Ile Asp Asp Leu Glu Leu Thr 945 950 955 960 Leu Ala Lys Val Glu LysGlu Lys His Ala Thr Glu Asn Lys Val Lys 965 970 975 Asn Leu Thr Glu GluMet Ala Gly Leu Asp Glu Ile Ile Ala Lys Leu 980 985 990 Thr Lys Glu LysLys Ala Leu Gln Glu Ala His Gln Gln Ala Leu Asp 995 1000 1005 Asp LeuGln Ala Glu Glu Asp Lys Val Asn Thr Leu Thr Lys Ala Lys 1010 1015 1020Val Lys Leu Glu Gln Gln Val Asp Asp Leu Glu Gly Ser Leu Glu Gln 10251030 1035 1040 Glu Lys Lys Val Arg Met Asp Leu Glu Arg Ala Lys Arg LysLeu Glu 1045 1050 1055 Gly Asp Leu Lys Leu Thr Gln Glu Ser Ile Met AspLeu Glu Asn Asp 1060 1065 1070 Lys Gln Gln Leu Asp Glu Arg Leu Lys LysLys Asp Phe Glu Leu Asn 1075 1080 1085 Ala Leu Asn Ala Arg Ile Glu AspGlu Gln Ala Leu Gly Ser Gln Leu 1090 1095 1100 Gln Lys Lys Leu Lys GluLeu Gln Ala Arg Ile Glu Glu Leu Glu Glu 1105 1110 1115 1120 2 13 PRTHomo sapiens 2 Asp Pro Leu Asn Glu Thr Val Val Gly Leu Tyr Gln Lys 1 510 3 13 PRT Homo sapiens 3 Asp Pro Leu Asn Glu Thr Val Met Gly Leu TyrGln Lys 1 5 10

What is claimed is:
 1. A method for the medical-diagnostic analysis ofthe expression and incorporation and of the ratio of wild type to mutantin different tissue and organ regions by comparing a deviation, causedby a mutation, of the amino acid composition of a protein to be examinedexpressed in the region of the mutation with a corresponding proteinwhich is expressed by a wild type lacking the mutation, wherein a sampleis taken from the organism at a site where the protein to be examined isexpressed, is detected, and/or plays a physiological role; either theprotein is concentrated or purified by methods of protein analysis,followed by a determination of its molecular weight, or a determinationof the molecular weight of the protein to be examined is performedwithout a pretreatment of the sample; and the ratio of expression andincorporation of wild type to mutant is quantified.
 2. The methodaccording to claim 1, characterized in that the protein to be examinedis cleaved into protein fragments.
 3. The method according to claim 1,characterized in that the protein to be examined is the heavy chain ofthe β-isoform of myosin or a fragment thereof obtainable withendopeptidase Lys-C.
 4. The method according to claim 1, characterizedin that determining the molecular weight is effected by means of directliquid chromatography-mass spectrometry coupling or indirectmatrix-assisted laser desorption ionization mass spectrometry.
 5. Themethod according to claim 1, for the medical-diagnostic quantificationof the expression and incorporation of wild type and mutant inphysiologically functional structures on the protein level.
 6. Themethod according to claim 1, for the medical-diagnostic detection of asyet unknown pathogenic and non-pathogenic mutations directly on theprotein level.
 7. The method according to claim 1, wherein the ratio ofexpression and incorporation of wild type to mutant is quantified bycapillary zone electrophoresis.
 8. The method according to claim 7,characterized in that the protein to be examined is the cause for adisease that is caused by a heterozygote point mutation in thecorresponding gene for that protein.